Safety and efficacy of mesenchymal stem cells in COVID‐19 patients: A systematic review and meta‐analysis

Abstract Background Coronavirus disease‐19 (COVID‐19) is a zoonotic disease that has become a global pandemic. The fast evolution of the COVID‐19 pandemic and persist problems make COVID‐19 highly infectious; publicly accessible literature and other sources of information continue to expand in volume. The mesenchymal stem cells (MSCs) therapy efficacy for COVID‐19 is debatable. Objective This systematic review and meta‐analysis (SRMA) aimed to evaluate the usefulness of MSCs in treating COVID‐19. Methods Relevant publications were retrieved from databases up to April 30, 2022. In the case of dichotomous data, the 95% confidence intervals (CIs) and pooled risk ratio (RR) were estimated with a random effects model (REM) or fixed effects model (FEM). The pooled mean difference (MD) and 95% CIs were calculated with REM or FEM in continuous data. In the outcomes, studies with insufficient or unusable data were reported descriptively. Results A total of eight randomized controlled trials (RCTs) with 464 people were chosen for this SRMA. Relative to the control group, mortality was significantly lower in the MSCs group (RR: 0.66, 95% CI: 0.44, 0.99, Z = 2.01, p = .04); other secondary outcomes, such as the clinical symptom improvement rate improved in the MSCs group (RR: 1.44, 95% CI: 1.05, 1.99, Z = 2.24, p = .03), clinical symptom improvement time (MD: −4.01, 95% CI: −6.33, −1.68, Z = 3.38, p = .0007), C‐reactive protein (CRP) (MD: −39.16, 95% CI: −44.39, −33.94, Z = 14.70, p < .00001) and days to hospital discharge (MD: −3.83, 95% CI: −6.19, −1.48, Z = 3.19, p = .001) reduced significantly in MSCs group. However, the adverse reaction incidence did not change significantly. Conclusions MSCs are a viable therapy option for COVID‐19 because of their safety and potential efficacy. With no significant adverse effects, MSCs can reduce mortality, clinical symptom improvement time, and days to hospital discharge, improve clinical symptoms, and reduce inflammatory cytokines CRP in COVID‐19. However, further high‐quality clinical studies are required to confirm these results.


| INTRODUCTION
At the stroke of the New Year 2020, coronavirus disease-19 (COVID-19), a zoonotic disease, turned into a global pandemic. 1COVID-19 is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 2It has been demonstrated that SARS-CoV-2 stimulates inflammasomes indirectly or directly, causing pyroptosis and, ultimately, tissue damage and a broad range of clinical symptoms. 3COVID-19 severity must be considered while developing a treatment plan.5][6] Although the COVID-19 situation is improving, many people may still have contracted the disease and are undergoing treatment.8][9] Therefore, in the event of a second outbreak, finding highly effective treatments for COVID-19 remains extremely important.][12] Recently, one of the most potent therapeutic approaches for COVID-19 symptoms and complications has been the use of mesenchymal stem cells (MSCs).Moreover, MSCs may help alleviate the long-term effects of COVID-19.In particular, MSCs are lauded for their immunomodulatory properties.MSCs were evaluated in treating several diseases. 13Human bone marrow cell suspensions were screened to identify MSCs that have lost their hematopoietic potential and form fibroblast cells like proliferating adherent colonies with the ability to differentiate into osteocytes, chondrocytes, and adipocyts both in vivo and in vitro. 14Multiple effector functions were modulated when MSCs interacted with immune cells from the adaptive and innate immune systems.MSCs establish peripheral tolerance, migrate to injured tissues, and inhibit proinflammatory cytokine production, promoting damaged cell survival when administered in vivo. 15They have substantial benefits for organs associated with COVID-19. 16Previous research has shown that MSCs do not express the SARS-CoV-2 spike (S) protein-recognizing and binding enzymes angiotensin-converting enzyme 2 (ACE2) or transmembrane serine protease 2 (TMPRSS2).The S protein is crucial to viral infection and transmission. 17,18herefore, MSCs are immune to SARS-CoV-2, 19 and treating COVID-19 with MSCs might be a successful strategy.
The evolving COVID-19 pandemic combined with ongoing issues means that systematic review and metaanalysis are very important.This SRMA will describe the reported performance of MSCs in treating novel coronavirus pneumonia.

| Inclusion and exclusion criteria
The following were the inclusion criteria for studies 20 : (1) include COVID-19 diagnosed patients; (2) only published types of randomized controlled trials (RCTs) were selected; (3) MSCs treatment was the intervention used; (4) the MSCs treatment (intervention arm) was compared with conventional care (control arm); (5) comprehensive information on both groups.Case reports, case series, and review articles were excluded.

| Search strategy and study selection
Our SRMA adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines for such studies. 21The China National Knowledge Infrastructure, the Cochrane Central Register of Controlled Trials (Central), Embase, the Web of Science, and Pubmed, were searched for any mention of COVID-19 and MSCs until April 30, 2022.Two authors independently checked the abstracts of each article to determine its eligibility.

| Study outcome measures
Mortality is the primary outcome, including all deaths that occur between randomization and the endpoint of clinical monitoring.Secondary outcomes include: (1) clinical symptom improvement, including the clinical improvement rate and clinical symptom improvement time; (2) C-reactive protein (CRP) degree of improvement; (3) incidence of adverse reactions was described as causing pain or discomfort that was not the treatment's intended aim; (4) days to hospital discharge measured from the time of admission till release.

| Literature screening, data extraction
The articles that we gathered were managed using Endnote X9.Two authors working independently selected articles no longer supported by the literature searches.After reading the abstract, a full-text analysis was conducted to check if it met the inclusion criteria.A third author was responsible for resolving any research selection disputes.The arbitrator addressed discrepancies between the data extractions by two independent assessors using a standard data extraction form. 20First author, sample size, design, year of publication, country, the intervention of patients in the group (treatment and control group), MSCs dose, and outcomes were the main elements of the extracted data.

| Risk of bias (ROB) assessment
The ROB was evaluated using the method described in the Cochrane Handbook for Systematic Reviews of Interventions. 20Eligible RCTs had their methodological quality assessed independently.The six types of biases are as follows: (1) Selection bias; (2) Implementation bias; (3) Measurement bias; (4) Follow-up bias; (5) Reporting bias; (6) Other biases: unbalanced baseline, insufficient sample size, conflict of interest.Each submission was rated as either "low risk," "unclear risk," or "high risk".

| Statistical analysis
Stata 16 and RevMan 5.4 were used to conduct the statistical analysis.We utilized pooled data if there were two or more similar studies available. 20The risk ratio (RR), 95% confidence interval (CI), and p values for dichotomous outcomes were determined.The MD, p values, and 95% CI were used for continuous variables.The statistical measure of heterogeneity, I 2 , was used for this analysis.I 2 < 50% and p ≥ .1 indicated that no heterogeneity was present, and I 2 ≥ 50% or p < .1 indicated heterogeneity. 20Sensitivity analysis was performed to find the cause of heterogeneity.Publication bias was evaluated using Egger's Test, with a cutoff of p < .05deemed statistically significant. 20| RESULTS

| Literature selection
After reviewing the abstracts and titles of the 1354 records obtained by the search strategy, 51 publications were retrieved and analyzed.Only eight RCTs met the criteria for inclusion.Simultaneously, the other articles were ruled out due to their absence of a control group, lack of a randomized controlled trial design, or their status as retrospective studies.Figure 1 is a flowchart depicting the article screening procedure.

| ROB within studies
Figure 2 depicts the bias evaluation, while Figure 3 displays the risk summary.Six of the eight RCTs included in this analysis employed computer-generated randomization, which was considered a low-risk type. 22,23,25,26,28,29he remaining two RCTs were considered to have unclear bias risk because they did not adequately describe their randomization methods. 24,27Six studies fully discussed keeping the distribution scheme concealed; this was deemed low-risk. 22,23,25,26,28,29The allocation concealing procedure was not described in the remaining two investigations, which was considered to have an unclear bias risk. 24,27The six studies were concealed using the following methods: (1) Randomized block design treatment assignment 22,23 ; (2) Randomization table generated by the R program 25,26 ; (3) Using SAS software 28 ; (4) Using a computerized random number generator. 29High risks of detection deviation and performance deviation were found in the three RCTs. 22,24,27All eight RCTs were deemed low risk regarding selective reporting of results and data integrity.None of the eight RCTs involved unbalanced baselines, small sample sizes, or conflicts of interest.

| Clinical improvement rate
Clinical symptom improvement rate There was substantial statistical heterogeneity across the two RCTs 24,27 that reported improvement of clinical symptoms (I 2 = 54%, Q test p = .14,Figure 5); thus, REM was used.Compared with the control group, MSCs showed significantly better results (RR: 1.44, 95% CI: 1.05, 1.99, Z = 2.24, p = .03,Figure 5).This proved that patients treated with MSCs improved their clinical symptoms at a rate 1.44 times greater than those receiving standard treatment or a placebo.Clinical evidence suggests that MSCs may aid COVID-19 patients with symptom improvement.
Clinical symptom improvement time Two RCTs 24,27 assessed clinical symptom improvement time and no heterogeneity was found among these studies (I 2 = 0%, Q test p = .46,Figure 6).The FEM was employed, clinical symptom improvement time was substantially shorter in the MSCs group compared with the standard care or placebo group (MD: −4.01, 95% CI: −6.33, −1.68, Z = 3.38, p = .0007,Figure 6), suggesting that MSCs is effective in reducing clinical symptom improvement time in patients with COVID-19.

| Days to hospital discharge
There was no significant heterogeneity across the two RCTs 24,27 regarding the number of days until patients were discharged from the hospital after treatment (I 2 = 0%, Q test p = .84,Figure 10).The FEM was employed.There was a significant difference between the MSCs group and the control group (MD: −3.83, 95% CI: −6.19, −1.48, Z = 3.19, p = .001,Figure 10), suggesting that patients receiving MSCs were able to leave the hospital sooner than those getting standard care or placebo.

| Publication bias detection
Then, we evaluated meta-analyses of mortality and adverse effects to see whether they were affected by publication bias.There was no publication bias in terms of mortality and incidence of adverse reactions (p > .05; Figure 11A, B); however, the publication bias for other outcomes could not be conducted due to the small number of included publications.

| DISCUSSION
Although the COVID-19 pandemic is getting better, we still need to seek effective methods for treating COVID-19 to better respond to this disease in future outbreaks.Thus, research on MSC treatment for COVID-19 remains significant and worth further exploration.COVID-19 directly leads to the disordering of adaptive and innate immune responses. 6,30Massive vaccination campaigns and antiviral treatment have been implemented, but reinfection and vaccine breakthough instances still occur; thus, researchers have focused on immunotherapies that attempt to lessen the severity of pathological changes in affected organs.In response to various chemokines, MSCs may be recruited to areas of inflammation, where they can regulate the activities of various immunocytes. 31Reducing the production of proinflammatory cytokines is one effect of MSCs' ability to influence host innate and adaptive immune responses. 32,33Because of their ability to suppress an overactive inflammatory response, MSCs may be a helpful therapeutic alternative for COVID-19 patients, especially those in urgent or life-threatening situations due to lung injury.In fact, from basic studies through human clinical trials, MSC therapy has been a staple of cell-based treatment. 34Multiple clinical trials using intravenous, intratracheal, and inhalation/nebulization administrations of MSC-based therapeutics are currently registered. 35The sources of MSCs are adipose tissue (fat) and bone marrow.Adipose-derived mesenchymal stem cells (AD-MSCs) are gaining a lot of attention in the medical field because of their abundant, easy isolation, and potential for various therapeutic applications.The stromal vascular fraction cells (SVFs) provide a rich source of AD-MSCs. 36SVFs and AD-MSCs present antiinflammatory, immune-modulatory, and proangiogenic activities. 37There are some potential uses for AD-MSCs and SVFs: tissue regeneration and repair and wound healing, autoimmune and inflammatory diseases, cosmetics and beauty apps, cardiovascular disease, neurological Disorders, Hair disease. 38,39Acute respiratory distress syndrome (ARDS) patients have also benefited from MSCs treatment. 40esults from this meta-analysis suggest that MSCs treatment may have a therapeutic impact on COVID-  response to cytokine production. 41Regarding clinical manifestation, there was a distinct improvement in clinical symptom improvement time and clinical symptom improvement.These results provide credence to using MSCs therapy to deal with COVID-19.Furthermore, the results demonstrated that MSCs were generally well-tolerated by patients and that MSCs therapy was safe, with only minimal side effects.
Patients with COVID-19 may benefit from MSCs because of their ability to modulate immunity, decrease proinflammatory cytokines, increase plasma antibodies against SARS-CoV-2, and promote lung  damage repair. 42,43Preclinical and clinical studies have demonstrated that MSCs treatment increases the survival rate of individuals with H7N9 influenza. 44These results are consistent with those of related research.
There is a dearth of literature on using MSCs in treating COVID-19 in meta-analysis.This meta-analysis has the benefit of including the most RCTs on MSCs therapy for COVID-19 compared with other published material.All eight publications selected were highquality RCTs that did not cherry-pick their findings and included the complete outcome markers.However, we recognize that there are limitations to our study.First, there is a wide range in sample size, degree of bias, and external validity of the included research.Second, there is a possibility that not all reports on MSC therapy for COVID-19 may have been included due to the limits of database searches and manual retrieval.Third, there was not enough time between the first publication and the follow-up study in any of the included articles to observe any long-term effects.As more evidence becomes available, we will update the literature review.Finally, larger multicohort RCTs with long-term follow-up are needed to determine the effectiveness of MSCs treatment in preventing pulmonary fibrosis.

F I G U R E 1
Flow diagram of the number of studies screened and included in the meta-analysis.T A B L E 1 Characteristics of the included randomized controlled trials.

F I G U R E 2
Risk of bias graph: review authors' judgments about each risk of bias item presented as percentages across all included studies.F I G U R E 3 Risk of bias summary: review authors' judgments about each risk of bias item for each included study.
19.In terms of sample size and variety of testing indicators, the current meta-analysis is the largest RCT research meta-analyzed of MSCs on COVID-19 outcome indicators.It evaluated eight RCTs, including MSCs, for the treatment of COVID-19 providing unambiguous evidence for the efficacy of MSCs.These findings support the hypothesis that MSCs may reduce mortality and hospital stays.Mortality rates were much lower in the MSCs group compared with the control group.CRP was significantly reduced in the MSCs therapy group.CRP is a sensitive biomarker for inflammation and host

F I G U R E 4
Forest plot of mesenchymal stem cells (MSCs) group and control group with the mortality.F I G U R E 5 Forest plot of mesenchymal stem cells (MSCs) group and control group with the clinical symptom improvement rate.F I G U R E 6 Forest plot of mesenchymal stem cells (MSCs) group and control group with the clinical symptom improvement time.F I G U R E 7 Forest plot of mesenchymal stem cells (MSCs) group and control group with the C-reactive protein (CRP).

F I G U R E 8
Sensitivity analysis of mesenchymal stem cells (MSCs) group and control group with incidence of adverse reactions.FI G U R E 9 Forest plot of mesenchymal stem cells (MSCs) group and control group with the incidence of adverse reactions.

F
I G U R E 10 Forest plot of mesenchymal stem cells (MSCs) group and control group with the days to hospital discharge.